The biogenesis of viral mRNA in African green monkey kidney cells during lytic infection with Simian virus 40 has been undertaken to examine gene expression in mammalian cells. In one approach, transcriptional mapping of cytoplasmic and nuclear RNA was performed. The results from this study showed that cytoplasmic RNA molecules contain variable RNA segments spliced to the main coding sequences, whereas a large fraction of nuclear RNA consists of unspliced molecules containing the intervening sequences. In a second approach, deletion mutants of SV40 have been constructed and RNA synthesis characterized. These experiments indicate that DNA sequences at or near the junctions of the splices serve as signals which control the biosynthesis of messenger RNAs.